ES2397293T3 - The use of alkaline phosphatase in the treatment of reduced renal function - Google Patents

Abstract

Use of alkaline phosphatase (FAL) in the manufacture of a medicament to improve reduced renal function, in which said renal function is reduced due to renal insufficiency

Description

The use of alkaline phosphatase in the treatment of reduced renal function

Description

The invention relates to the field of medicine and, in particular, to the use of alkaline phosphatase in the treatment of kidney diseases such as reduced renal function. The present invention also relates to the field of pharmacy and, in particular, to the pharmaceutical use of alkaline phosphatase.

There are multiple kidney diseases that can cause reduced kidney function. Acute renal failure (ARF) is one of the renal disorders that causes reduced renal function. Without limiting the scope of the invention, the IRA is described in more detail.

Acute renal failure (ARF) is defined as an acute loss of renal function that causes an increase in the level of serum creatinine.

The annual incidence of ARI acquired in the community is approximately 100 cases per million inhabitants and, at present, it is only diagnosed in 1% of hospital admissions. On the other hand, IRA acquired in hospitals occurs in up to 4% of hospitalizations and 20% of intensive care admissions. This increase in the incidence of acquired ARF at the hospital level is multifactorial: it is related to an aging population with a higher risk of ARF, the high prevalence of possible nephrotoxic exposures in the hospital environment and the increasing severity of the disease.

In the last 40 years, the survival rate for acute renal failure has not improved, mainly because the affected patients are now older and have more comorbid conditions. Depending on the severity of renal failure, the mortality rate can vary from 7 percent to as much as 80 percent.

In acute renal failure, the glomerular filtration rate decreases for days to weeks. As a result, the excretion of nitrogen waste is reduced, and the balance between fluids and electrolytes cannot be maintained. Patients with acute renal failure do not usually have symptoms, and the condition is diagnosed by observing elevations in blood urea nitrogen (NUS) and serum creatinine levels. Complete renal arrest occurs when the serum creatinine level increases by at least 0.5 mg per dl per day (equivalent to 44 μmol per liter per day) and urine output is less than 400 ml per day (oliguria ).

Pathophysiology: The driving force of glomerular filtration is the pressure gradient from the glomerulus into Bowman's space. Glomerular pressure depends primarily on renal blood flow (FSR) and is controlled by combining the resistance of afferent and efferent renal arterioles. Regardless of the cause of ARF, reductions in the FSR represent a common pathological pathway to decrease glomerular filtration rate (GFR). The etiology of the IRA comprises 3 main mechanisms.

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Prerenal insufficiency is defined by conditions with normal tubular and glomerular function. In acute prerenal renal failure, the problem is in the deterioration of renal blood flow as a result of a true decrease in intravascular volume, the decrease in effective circulation volume to the kidneys or agents that alter renal blood flow as a result of the decrease of the TFG.

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Intrinsic acute renal failure is divided into 4 categories: tubular disease, glomerular disease, vascular disease and interstitial disease. In intrinsic acute renal failure, the renal parenchyma is damaged.

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Postobstructive renal failure initially causes an increase in tubular pressure, decreasing the driving force of the filtration. Postrenal acute renal failure can only occur if both outflow urinary tracts are obstructed or the outflow tract of a single kidney is obstructed. Most commonly, the condition is due to obstruction of the lower urinary tract.

Prerenal insufficiency

The main agents that cause acute prerenal renal failure are angiotensin converting enzyme (ACE) inhibitors and non-steroidal anti-inflammatory drugs (NSAIDs). ACE inhibition prevents the conversion of angiotensin I to angiotensin II, which leads to decreased levels of angiotensin II. Angiotensin II increases the glomerular filtration rate by causing constriction of the efferent arteriole and its absence decreases the glomerular filtration rate due to dilation of the efferent arteriole.

Intrinsic Acute Kidney Failure

Tubular disease

Acute tubular necrosis caused by the decrease in FSR is the most common cause of intrinsic acute renal failure in hospitalized patients. The decrease in renal blood flow causes ischemia in the renal parenchyma. If the ischemia is prolonged, acute tubular necrosis may develop. This initial ischemic attack triggers the production of oxygen free radicals and enzymes that continue to cause cell damage, even after the restoration of FSR. Tubular cell damage breaks the solid junctions between cells, allowing glomerular filtration leaks to occur again and a greater decrease in effective GFR. In addition, the dying cells detach in the tubules, forming clogging cylinders, which further reduce GFR and cause oliguria. Acute ischemic tubular necrosis is usually reversible, but if the ischemia is severe enough to cause cortical necrosis, irreversible renal failure can occur.

Acute tubular necrosis has three phases. Kidney damage develops during the initial phase, which lasts from hours to days. In the maintenance phase, which lasts from days to weeks, the glomerular filtration rate reaches its lowest point and urine production is at its lowest level. The recovery phase lasts for days, often beginning with diuresis due to post-tubular tubular necrosis. The hypovolemia produced as a result of an overproduction of urine is a problem that appears in this phase. Despite the recovery of urine production, patients may continue to have difficulty with uremia and homeostasis of electrolytes and acid, because tubular function is not fully recovered.

Glomerular disease

The most common glomerular disease that produces ARF is glomerulonephritis. Glomerulonephritis is characterized by hypertension, proteinuria and hematuria. Of the many types of glomerulonephritis, most are associated with chronic kidney disease. In general, the two types of glomerulonephritis that cause acute renal failure are rapidly progressive glomerulonephritis and acute proliferative glomerulonephritis. The latter type occurs in patients with bacterial endocarditis or other post-infectious conditions.

Vascular disease

Microvascular or macrovascular disease (occlusion of the main renal arteries or severe abdominal aortic disease) can cause acute renal failure.

Interstitial disease

Acute interstitial nephritis usually presents with fever, rash and eosinophilia. Urine staining that is positive in eosinophils suggests the presence of this condition. Acute interstitial nephritis is usually the result of an allergic reaction to a drug, but it can also be caused by an autoimmune disease, an infection or an infiltrative disease.

The recovery of the IRA depends primarily on the restoration of the FSR. Early normalization of FSR predicts a better prognosis for recovery of renal function. In prerenal insufficiency, the restoration of circulating blood volume is usually sufficient. Rapid relief of urinary obstruction in postrenal insufficiency causes a rapid decrease in vasoconstriction. With intrinsic renal failure, only the restoration of blood volume does not restore renal function. The elimination of tubular toxins and the initiation of therapy for glomerular or tubular diseases decreases renal afferent vasoconstriction and may reverse ARF. The initial treatment should focus on correcting the balance between fluid and electrolytes, and uremia, while looking for the cause of acute renal failure. A patient with decreased volume is resuscitated with saline. However, it is more common for volume overload to occur, especially if the patients are oliguric or anuric.

An example of a current treatment is furosemide (Lasix) administered intravenously. Another example of one of the current treatments is calcium administered intravenously. Potassium can be temporarily moved to the intracellular compartment using intravenously administered insulin (10 units) and glucose (25 g), inhaled beta agonists or intravenously administered sodium bicarbonate. Potassium excretion is achieved with sodium polystyrene sulfonate (Kayexalate) and / or diuretics. Sodium polystyrene sulphonate is administered orally (from 25 to 50 g mixed with 100 ml of 20 percent sorbitol) or in the form of an enema (50 g in 50 ml of 70 percent sorbitol and 150 ml of running water ). If these measures do not control the level of potassium, dialysis begins.

Acidosis is commonly treated with sodium bicarbonate intravenously or orally if the level of serum bicarbonate is less than 15 mEq per l (15 mmol per liter) or the pH is less than 7.2. Patients can also be treated orally with sodium bicarbonate tablets, Shohl's solution in doses of 30 ml or baking soda powder. Serum bicarbonate levels and pH should be strictly controlled. Intractable acidosis requires dialysis.

All medications should be reviewed, and doses should be adjusted based on the glomerular filtration rate and serum levels of the medications.

Between 20 and 60 percent of patients require short-term dialysis, particularly when the NUS exceeds 100 mg per dl (35.7 mmol per l of urea) and the serum creatinine level exceeds the range of 5 to 10 mg per dl (442 to 884 μmol per l). Indications for dialysis include acidosis and electrolyte abnormalities that do not respond to drug therapy, fluid overload that does not respond to diuretics and uremia. In patients with progressive acute renal failure, the indication is to urgently consult a nephrologist.

In WO 2005/074978, the use of alkaline phosphatase for the prophylaxis or treatment of LPS-mediated diseases is disclosed. According to WO 2005/074978, alkaline phosphatase acts by detoxifying the rest of lipid A of the LPS. LPS-mediated kidney diseases are among the diseases that can be treated with alkaline phosphatase.

Part of the aforementioned treatments restore (partially) renal function. Another treatment improves function compared to people who do not receive treatment. However, alternative treatments are still needed.

The objective of the present invention is to provide an alternative treatment to improve renal function, especially in cases where renal function is impaired / reduced, at least partially.

The present invention provides an alternative treatment in which alkaline phosphatase is used to improve a situation in which renal function is reduced.

In a first embodiment, the invention provides the use of alkaline phosphatase (FAL) in the manufacture of a medicament for improving reduced renal function, wherein said renal function is reduced due to renal insufficiency.

The term "improve" includes slowing down the reduction, stopping the reduction, or at least partially reversing the reduced renal function.

The term "reduced renal function" is normally used to refer to a situation in which renal function is reduced compared to the value of at least one related renal parameter to a recognized or average value (laboratory) of said parameter. If, for example, the amount of protein present in the urine of a subject (preferably, of a human being) is (significantly) greater than a recognized value or average (laboratory) value, said person is said to have a "renal function" reduced. " The corresponding analysis can be performed in a laboratory, but also in the domestic environment. For example, since September 2006, the Dutch company "Nierstichting" has introduced a simple analysis test (called Niercheck) that can be done at home to check if the kidneys are working properly. This test analyzes the amount of urine protein.

Other examples of parameters that can be analyzed are glomerular filtration rate (GFR), serum creatinine levels, electrolyte disturbance, the amount of urine produced, blood urea nitrogen (NUS), calcium, phosphorus, albumin levels or red and white blood cells in the urine. Other tests that can be performed are a complete blood count with differential. Preferably, all possible patients undergo the following urinalysis: test with test strip, microscopy, sodium and creatinine levels, and determination of urinary osmolality.

As described herein within the experimental section, it is also possible to analyze the presence or absence of an RNA molecule from a urine sample. Preferably, said RNA molecule is an mRNA molecule. Even more preferably, said mRNA molecule is iNOS mRNA. In a more preferred embodiment, said RNA is obtained from renal cells secreted in the urine.

Based on the analysis described above, it is determined whether a subject (preferably, a human being) is suffering from renal renal function or not. A decrease in GFR or an elevated serum creatinine level or a reduction in the amount of urine produced or any combination thereof are, for example, (a) clear indications that the subject analyzed needs treatment as described herein.

Thus, the use described herein may be preceded by an analysis step comprising, for example, taking a sample of a subject suspected of having reduced renal function and analyzing said sample for any of the parameters. previously given (or a combination thereof) and compare the result (s) obtained with the average or recognized values. Examples of suitable samples are a urine sample or a blood sample. The analysis of urine and / or blood samples may also be accompanied by a renal ultrasound examination. In a preferred embodiment, the analysis is performed under the responsibility of a doctor. As soon as it is clear that renal function has decreased, treatment according to the invention is initiated.

A reduced renal function may occur as a result of different disorders. Many cases of impaired renal function cause a mild and asymptomatic disease that is not recognized by the patient, that does not call the attention of doctors and that is not diagnosed. The incidence and prevalence of such mild episodes of renal function impairment are unknown, but are considered to be important. However, such mild dysfunction can lead to serious problems and, therefore, early treatment is important (for example, as described herein).

The invention provides the use of alkaline phosphatase (FAL) in the manufacture of a medicament for improving reduced renal function, wherein said renal function is reduced due to renal insufficiency. Kidney failure is the condition in which the kidneys stop functioning properly. Physiologically, renal failure is described as a decrease in the glomerular filtration rate. Clinically, this manifests itself in an elevated serum creatinine level. In general, it can be divided into two categories: acute renal failure and chronic renal failure.

Chronic renal failure (CRF) develops slowly and initially gives few symptoms. It can be the complication of a large number of kidney diseases such as glomerulonephritis, chronic pyelonephritis and urinary retention. End-stage renal failure (IRFT) is the last consequence, in which case, dialysis is usually required until a donor is found to allow a kidney transplant. In acute renal failure, kidney function is almost completely inhibited. In a preferred embodiment, said renal failure is acute renal failure.

Acute renal failure (ARF) is, as the name implies, a rapid progressive loss of renal function that is usually characterized by oliguria (decreased urine output, quantified as less than 400 ml per day in adults, less than 0.5 ml / kg / h in children or less than 1 ml / kg / h in babies), disturbances of body water and body fluids, and electrolyte disorder. Patients who suffer from acute renal failure are usually hospitalized due to the severity of their condition. Currently, an underlying cause must be identified to stop progress, and dialysis may be necessary until these root causes can be treated. Based on the present invention, alkaline phosphatase therapy can be started immediately even without knowing the underlying cause and without wasting precious time. In a preferred embodiment, the invention provides the use of alkaline phosphatase (FAL) in the manufacture of a medicament for improving reduced renal function, wherein said reduced renal function is induced or maintained or aggravated due to renal insufficiency and in which renal failure is acute renal failure; that is, in a preferred embodiment, the invention provides the use of alkaline phosphatase (FAL) in the manufacture of a medicament for treating reduced renal function induced or maintained or aggravated by acute renal failure (ARF).

Acute renal failure may be present in addition to chronic renal failure. This is called acute renal failure over chronic. The acute part of acute chronic renal failure can be reversible and the goal of treatment is to return the patient to their initial renal function, which is commonly measured by serum creatinine. In yet another embodiment, the invention therefore provides the use of alkaline phosphatase (FAL) in the manufacture of a medicament for improving reduced renal function, in which said renal function is reduced (in addition) due to renal insufficiency. acute over chronic.

In yet another preferred embodiment, the reduced renal function is reversible, that is, the renal function can be improved, at least somewhat, by the treatment according to the invention.

As Agrawal and Swartz describe in detail (American Family Physician, April 1, 2000, vol. 61, No. 7, cover story), the cause of acute renal failure can be determined through the use of a phased approach. Based on this article, the expert can easily divide patients into any of the 3 categories of ARI, that is, determine if a patient suffers from prerenal ARI, intrinsic ARI or postrenal ARF.

For example, the diagnosis of intrinsic ARF is commonly determined based on the background and physical observations that indicate it, fractional excretion of sodium of more than 3%, urea osmolality of 250 to 300 mOsm and active ureic sediment . In acute intrinsic ARF, the renal parenchyma is damaged. The damage of the tubular cells leads to certain microscopic observations in the urine. The parenchymal lesion affects the reabsorption of sodium and generates the parameters described above, such as a fractional excretion of sodium of more than 3 percent and an isotonic urine osmolality of 250 to 300 mOsm. Intrinsic acute renal failure is divided into 4 categories: tubular disease, glomerular disease, vascular disease and interstitial disease.

Acute tubular necrosis is the most common cause of intrinsic acute renal failure in hospitalized patients. This condition is usually caused by ischemia or toxins. In ischemic acute tubular necrosis, unlike acute prerenal renal failure, the glomerular filtration rate does not improve with the restoration of renal blood flow. Acute ischemic tubular necrosis is usually reversible, but if the ischemia is severe enough to cause cortical necrosis, irreversible renal failure can occur. Contrast agents and antibiotics, especially aminoglycosides, are the agents most frequently associated with acute tubular necrosis. The condition may also be caused by the myoglobinuria pigment (rhabdomyolysis) or hemoglobinuria (hemolysis). Acute tubular necrosis has three phases. Kidney damage, which develops during the initial phase and lasts from hours to days. In the maintenance phase, which lasts from days to weeks, in which the glomerular filtration rate reaches its lowest point and urine production is at its lowest level. The recovery phase lasts for days, often beginning with post-acute tubular necrosis diuresis. The hypovolemia produced as a result of an overproduction of urine is a problem that occurs during this phase. Despite the recovery of urine production, patients may continue to have difficulty with uremia and homeostasis of electrolytes and acid, because tubular function is not fully recovered. Diligent surveillance is indicated in all phases of acute tubular necrosis. Patients at risk of acute tubular necrosis include those who have diabetes, congestive heart failure or chronic renal failure. Acute tubular necrosis can be prevented by early treatment of patients with reversible causes of acute prerenal or ischemic renal failure, and maintaining adequate hydration in patients receiving nephrotoxins. Once acute tubular necrosis develops, treatment is supportive. Drugs such as mannitol, loop diuretics, dopamine and calcium channel blockers have successfully enhanced diuresis in animals, but no similar results have been obtained in humans.

In a preferred embodiment, the invention provides the use of alkaline phosphatase (FAL) in the manufacture of a medicament for improving reduced renal function, wherein said renal function is reduced due to renal failure, preferably, acute renal failure, and wherein said reduced renal function is induced or maintained or aggravated by an intrinsic acute renal failure, preferably acute tubular necrosis. That is, the invention also provides the use of alkaline phosphatase (FAL) in the manufacture of a medicament for treating acute tubular necrosis.

Glomerulonephritis is characterized by hypertension, proteinuria and hematuria. Glomerulonephritis is commonly described as an inflammation of the glomeruli. Of the many types of glomerulonephritis, most are associated with chronic kidney disease. In general, the two types of glomerulonephritis that cause acute renal failure are rapidly progressive glomerulonephritis and acute proliferative glomerulonephritis. The latter type occurs in patients with bacterial endocarditis or other post-infectious conditions. Rapidly progressive glomerulonephritis may be a primary disorder, or it may be secondary to systemic disease. Once this condition is suspected, treatable systemic disease should be sought using serological markers or renal biopsy. Renal function can decrease rapidly in patients with rapidly progressive glomerulonephritis, and end-stage renal disease can develop in days to weeks. Patients with rapidly progressive glomerulonephritis are usually treated with glucocorticoids and cyclophosphamide (Cytoxan). It is believed that plasma exchange will benefit patients with Goodpasture syndrome, but it has not been proven to be beneficial in patients with other types of glomerulonephritis.

In addition, this type of ARI can be treated by the use of alkaline phosphatase and, therefore, in a preferred embodiment, the invention provides the use of alkaline phosphatase (FAL) in the manufacture of a medicament for improving reduced renal function, in that said renal function is reduced due to renal failure, preferably acute renal failure, and in which reduced renal function is induced or maintained or aggravated by intrinsic acute renal failure, preferably glomerulonephritis. That is, the invention also provides the use of alkaline phosphatase (FAL) in the manufacture of a medicament for treating glomerulonephritis.

Renal insufficiency, in general, and acute renal failure, in particular, can evolve due to different underlying causes or in the course of various diseases such as ischemia, contrast agent pigments, systemic lupus erythematosus, small vessel vasculitis, Henoch-Schönlein purpura, Goodpasture syndrome, encarditis, post-streptococcal infection, post-pneumococcal infection, diabetes, hypertension, atherosclerosis or cancer. It is also known that the use of certain antimicrobial agents (such as amphotericin B, caspofungin, vancomycin, levofloxacin and aminoglycosides such as tobramycin and gentamicin), other drugs (for example, chemotherapeutic agents (for example, cisplatin, carboplatin, methotrexate), inhibitors of protease (such as indinavir and ritonavir), gold, lithium, anti-inflammatory drugs (such as nonsteroidal anti-inflammatory drugs, cyclosporine, tacrolimus, sirolimus), blood pressure medications (such as angiotensin converting enzyme inhibitors ( ACE) and angiotensin receptor (BRA) blockers and certain chemicals (such as silicates, hydrocarbons, heavy metals (such as Cd, Hg, Pb), insecticides, herbicides, ethylene glycol and bacterial toxins (such as tetanus , streptococcal toxins)) cause reduced renal function in subjects who have taken or been exposed to such agents or produce chemical counts

The present invention can be used to treat reduced renal function induced or maintained or aggravated by any of the aforementioned causalities. Accordingly, in a preferred embodiment, the invention provides the use of alkaline phosphatase (FAL) in the manufacture of a medicament for improving reduced renal function, in which said renal function is reduced due to renal insufficiency and in which said Reduced renal function is induced or maintained or aggravated by a medication, a drug and / or a toxin, preferably selected from the group consisting of antibiotics, protease inhibitors, chemotherapeutic agents, anti-inflammatory agents, blood pressure medications, insecticides , herbicides, ethylene glycol, contrast dyes, heavy metals and bacterial toxins.

In yet another preferred embodiment, the invention provides the use of alkaline phosphatase (FAL) in the manufacture of a medicament for improving reduced renal function, in which the improvement of renal function obtained does not occur as a result of the detoxification of LPS by the FAL. In an even more preferred embodiment, the invention provides the use of alkaline phosphatase (FAL) in the manufacture of a medicament for improving reduced renal function, in which said renal function is reduced due to renal insufficiency, and in which said reduced renal function is induced or maintained or aggravated by the decrease in renal blood flow and / or ischemia. Said decrease in renal blood flow and / or ischemia, preferably, is induced or maintained or aggravated due to dehydration, heart failure, septic shock, severe blood loss, hypertension, atherosclerosis and / or thrombosis. In yet another preferred embodiment, the invention provides the use of alkaline phosphatase (FAL) in the manufacture of a medicament for improving reduced renal function, in which said renal function decreases due to renal insufficiency and in which said renal function reduced is induced or maintained or aggravated by the decrease in renal blood flow and / or ischemia, in which said decrease in renal blood flow and / or ischemia, preferably, is induced or maintained or aggravated by a drug or drug or toxin, preferably selected from the group consisting of anti-inflammatory agents (more preferably, non-steroidal anti-inflammatory drugs) and blood pressure medications (most preferably, angiotensin converting enzyme (ACE) inhibitors and / or receptor blockers of angiotensin (BRA)

The use of alkaline phosphatase (FAL) in the manufacture of a medicament to treat or prevent reduced renal function produced as a result of the increase in the expression of inducible NO synthase (iNOS) (renal) is also revealed. Such treatment is very useful in the treatment of so-called early renal damage or insufficiency and, therefore, can be used, for example, to treat or prevent early kidney disease. People in need of such treatment can be easily identified by determining the amount of iNOS and comparing the value obtained with a medium level. By administering an effective amount of alkaline phosphatase to a person diagnosed with damage or early renal failure, the amount of renal iNOS is reduced. The present inventors have determined that treatment with alkaline phosphatase produced an attenuation of the increase in the expression of iNOS mRNA in renal cells, generating a reduction in the urinary excretion of NO metabolites. FAL treatment improves inflammatory responses, producing a reduction in the induction of renal iNOS expression, which leads to an attenuation of NO metabolite production and reduced proximal tubular damage.

Renal insufficiency is usually accompanied by structural damage to renal cells that are secreted in the urine. Isolation of these cells from a urine sample and subsequent analysis of RNA synthesis provides a useful tool for controlling renal function, kidney damage and, over time, the reversal of damage. In Example 1, the expression of inducible nitric oxide synthase (iNOS) mRNA in renal cells secreted in the urine is used as a marker of renal failure and, therefore, could be used to control damage and inversion. of the damage thanks to a treatment with FAL. The underlying mechanism of damage is the induction of reactive oxygen species (ERO) by iNOS, leading to kidney leakage. The reduction of the induction of ERO through the infraregulation of the iNOS can be controlled by using this procedure.

Therefore, the use of FAL in the preparation of a medicament to improve reduced renal function is revealed, which further includes the analysis of the presence or absence of an RNA molecule in a urine sample. Preferably, said RNA molecule is an mRNA molecule. Even more preferably, said mRNA molecule is iNOS mRNA. Most preferably, said RNA is obtained from renal cells secreted in the urine.

The invention uses alkaline phosphatase (FAL) to improve a reduction (or a condition) of renal function.

Alkaline phosphatase (FAL); EC 3.1.3.1 according to the IUBMB enzyme nomenclature, whose common name is alkaline phosphatase (FAL), is an enzyme that catalyzes the reaction of a phosphatase and H2O monoester in an alcohol and phosphate. Another name / s of the FAL are alkaline phosphomonoesterase; phosphomonoesterase; glycerophosphatase; alkaline phosphohydrolase; alkaline phenyl phosphatase; orthophosphoric monoester phosphohydrolase (alkaline optimum). The systematic name of the FAL is phosphate monoester phosphohydrolase (alkaline optimum).

FAL is an enzyme of wide specificity; It also catalyzes the transphosphorylations. In humans and other mammals, at least four distinct, but related alkaline phosphatases are known. These are intestinal, placental, placental and hepatic / bone / renal (or non-tissue specific) alkaline phosphatase. The first three are found together on chromosome 2, while the non-specific shape of the tissue is located on chromosome 1. The exact physiological functions of FALs are unknown, but FAL seems to be involved in a large number of physiological processes. .

A source of FAL may be a commercial FAL enzyme or any composition comprising the FAL enzyme and any medium that is capable of producing a functional FAL enzyme in the context of the present invention, such as DNA or RNA nucleic acids encoding a FAL protein. . The nucleic acid encoding the FAL can be incorporated into suitable vectors such as plasmids, phagemids, phages, (retro) viruses, transposons, gene therapy vectors and other vectors capable of inducing or conferring the production of the FAL. Native or recombinant microorganisms such as bacteria, fungi, protozoa and yeasts can also be applied as a source of the FAL in the context of the present invention.

Compositions containing FAL for use according to the present invention preferably comprise a eukaryotic FAL, more preferably, a mammalian FAL, which may be of the non-tissue specific FAL type, such as liver-bone or renal, or specific of the tissue, such as placental FAL, intestinal FAL and placental FAL. The latter, also known as germ cell FAL, is found in the testes, thymus and certain germ cell tumors (1) and is closely related to both placental and intestinal alkaline phosphatase forms (2). The expert is perfectly trained to search nucleic acid banks and select a sequence that encodes alkaline phosphatase. Most preferably, the mammalian FAL is a human or bovine FAL. Accordingly, in a preferred embodiment, the invention provides the use of alkaline phosphatase (FAL) according to the invention, wherein said FAL is mammalian FAL and, even more preferably, wherein said FAL is human FAL. Non-restrictive examples of a human FAL sequence can be found in the NCBI (Genpept) collection and include: NP_001622 (intestinal FAL), NP_001623 (placental FAL), NP_112603 (placental type FAL) or NP_000469 (non-specific FAL of the tissue). The invention also comprises the use of a polymorphism of any of said sequences. In yet another preferred embodiment, said FAL is placental FAL, placenta type FAL, intestinal FAL or hepatic / bone / renal FAL.

In yet another preferred embodiment, the invention provides the use of the FAL according to the invention, wherein said FAL is recombinant FAL.

From an configuration point of view, an alkaline phosphatase consists of approximately two domains: a crown domain and an active site domain. The active site domain can be divided into separate parts, such as the catalytic residue and the three metal ion sites (Zn1, Zn2 and Mg3). From a primary structure point of view, it is clear that the crown domain is flanked by the amino acids that form the active site domain. The amino acid sequence of alkaline phosphatases and the relative positions of the catalytic domain and the crown domain are known to those skilled in the art. As an example, reference is made to Figure 10, which shows, among others, the amino acid sequence of the four human alkaline phosphatases. The crown domain is underlined in these sequences.

Alkaline phosphatases are present in almost all organisms, from bacteria to humans. In a preferred embodiment, the FAL is an isolated or recombinant alkaline phosphatase comprising a crown domain and a catalytic domain, wherein said crown domain and said catalytic domain are obtained from different alkaline phosphatases and which at least one of said Different phosphatases is a human phosphatase. The other phosphatase is, for example, alkaline phosphatase from Escherichia coli or one of the seven known bovine intestinal alkaline phosphatases. In a preferred embodiment, an isolated or recombinant alkaline phosphatase is used comprising a crown domain and a catalytic domain, wherein said crown domain and said catalytic domain are obtained from different alkaline phosphatases and in which the different alkaline phosphatases are human phosphatases This is especially useful if modified phosphatase is subsequently used in human therapy. It is expected that such (genetically) modified phosphatases of human origin are not or are very little immunogenic. However, it is clear to experts that if a modified phosphatase is used, for example, in "in vitro" or "ex vivo" diagnoses, it may be composed, for example, of a human alkaline phosphatase and one of E. coli or it may be composed of a bovine alkaline phosphatase or one of E. coli.

In yet another preferred embodiment, the FAL is an isolated or recombinant alkaline phosphatase comprising a crown domain and a catalytic domain, wherein said crown domain and said catalytic domain are obtained from different alkaline phosphatases and in which said domain of corona is the crown domain of placental FAL (ALPP) and in which said catalytic domain is the catalytic domain of intestinal FAL (ALPI). Preferably, at least one of said different phosphatases is a human phosphatase and, in an even more preferred embodiment, both

Different phosphatases are human phosphatases. Other preferred mutants with the changed domain that are based on human alkaline phosphatases are:

Catalytic domain

Crown domain Denominated

ALPI

GCAP catALPI / crownGCAP

TNAP

catALPI / crownTNAP

ALPP

GCAP catALPP / crownGCAP

TNAP

catALPP / crownTNAP

GCAP

ALPI catG CAP / crownALPI

ALPP

catGCAP / crownALPP

TNAP

catGCAP / crownTNAP

TNAP

ALPI catTNAP / crownALPI

ALPP

catTNAP / crownALPP

GCAP

catTNAP / crownGCAP

For the sake of clarity, ALPI is intestinal FAL, ALPP is placental FAL, GCAP is placental type FAL and TNAP is non-tissue specific FAL.

It is obvious that combinations can also be made between the catalytic domain of the alkaline phosphatase of Escherichia coli or any of the human forms (ALPI, ALPP, GCAP or TNAP) with the crown domain of an alkaline bovine intestinal alkaline phosphatase. In addition, combinations of the crown domain of a bovine intestinal alkaline phosphatase with the catalytic domain of the alkaline phosphatase of Escherichia coli or any of the human forms can also be produced.

Another class of useful modified phosphatases are phosphatases that under natural conditions are linked to the membrane of a cell through a glycosylphosphatidylinositol (GPI) anchor, but are now modified so that they are no longer bound to the membrane of a cell. Examples of phosphatases that are anchored through GPI are alkaline phosphatase and 5'-nucleotidase. All isoenzymes are functionally active in the cell membrane and deficient forms in the GPI anchor are not naturally present at detectable levels. Although the activity of serum alkaline phosphatase has been demonstrated, it is generally accepted that the enzyme is still present in the membrane fractions or detached membrane vesicles. FAL activity in milk is also present in fractions containing membrane vesicles. The GPI anchor is stored as a precursor molecule in the cell in which it is attached to the binding site through a transamidase. The central structure of the GPI anchor is identical in mammals, but modifications are known depending on the type of cell.

Alkaline phosphatases are predominantly in association with plasma membranes through their GPI anchor. For example, neutrophils present the enzyme against the bottom of their negatively charged cell membrane instead of releasing it in the inflammatory microenvironment. For this reason, prior to the present invention, it was generally recognized that for optimal in vivo activity of FAL, the envelope should be within a cell membrane or a vesicular membrane.

For a pharmaceutical use of FAL in human subjects, it is preferred, for most applications, to apply human forms of the enzyme to medications and treatment, since FAL forms obtained from other species can be immunogenic in humans and the treatment could cause immunological reactions and pathological side effects. In some subjects, even lethal side effects can occur, that is, an anaphylactic shock can be generated (shown in animal studies of the present inventors), therefore, it is preferable to minimize the risks of suffering immunological side effects. Since, in practice, the isolation of FAL from humans is unfeasible, human recombinant forms of FAL proteins can be produced in the usual manner on different recombinant expression platforms. However, the expression and purification of proteins modified in terms of GPI and membrane anchored is very difficult; GPI proteins are difficult to separate from membranes, and difficult to isolate and purify. However, before the present invention, the GPI anchor and the membrane location have always been considered essential for the biological activity of the FAL.

However, in one of the embodiments of the present invention, the FAL is an isolated or recombinant phosphatase comprising a modification in the glycosylphosphatidylinositol (GPI) signal sequence, in which said modification causes a secreted phosphatase, i.e., the phosphatase It is not bound to the cell membrane.

In a preferred embodiment, an isolated or recombinant phosphatase is used which comprises a modification in the glycosylphosphatidylinositol (GPI) signal sequence, wherein said modification causes a secreted phosphatase that is biologically active, that is, shows an activity towards a (determined ) biological substrate.

There is no general sequence responsible for the union of a GPI anchor, but there is a clear consensus:

1) hydrophobic stretch of amino acids at the C-terminal end (at least 11 amino acids, but preferably more than 11 amino acids);

2) upstream of the hydrophobic region, a spacer of hydrophilic amino acids (5-12 amino acids);

3) GPI is linked to a small amino acid: glycine, aspartic acid, asparagine, alanine, serine or cysteine;

4) The 2 subsequent amino acids downstream of the GPI binding site must be small amino acids and in most cases they are selected from glycine, aspartic acid, asparagine, alanine, serine or cysteine.

Based on this consensus, the person skilled in the art is able to mutate this consensus, for example, by inserting one or multiple amino acids and altering part of the consensus. However, in a preferred embodiment, an isolated or recombinant phosphatase is used which comprises a modification in the glycosylphosphatidylinositol (GPI) signal sequence, wherein said modification produces a secreted phosphatase and in which said modification comprises a mutation or a deletion of the amino acid sequence that encompasses the signal sequence of GPI consensus.

For applications in human therapy, it is desired that the resulting modified phosphatase is not or is very little immunogenic, that is, that the modified phosphatase is essentially of human origin. In a preferred embodiment, the FAL is an isolated or recombinant phosphatase comprising a modification in the glycosylphosphatidylinositol (GPI) signal sequence, wherein said modification produces a secreted phosphatase (preferably, with activity against a certain biological substrate) and in the that said phosphatase is a human phosphatase.

Examples of phosphatases having GPI anchoring are alkaline phosphatase and 5'-nucleotidase, and therefore, in a preferred embodiment, an isolated or recombinant phosphatase is used which comprises a modification in the signal sequence of glycosylphosphatidylinositol (GPI), in which said modification produces a secreted phosphatase and wherein said phosphatase is an alkaline phosphatase, for example, a human alkaline phosphatase such as, for example, human hepatic-renal-bone phosphatase, human intestinal alkaline phosphatase or alkaline placenta type phosphatase human

It is evident that any of the secretable modified phosphatases described can be produced, for example, by the introduction into a host cell of a nucleic acid capable of encoding said secretable phosphatase, preferably, operably linked to regulatory sequences, and allowing said host cell to express said secretable phosphatase and, optionally, by isolating the phosphatase produced from the medium in which the host cell is grown and / or maintained. However, apart from the mutations of the GPI binding sequence mentioned above, there are other methods that produce secreted proteins without GPI anchoring:

1) After expression as membrane-anchored proteins, phospholipases can be used to cleave the GPI anchor.

2) Interference in the production of the GPI anchor or the use of a cell (cell type) that is deficient in the production of GPI anchor can also be used to create a secretable form of a GPI anchored protein in another way. Examples of cell lines that have been created to be deficient in the biochemistry of the GPI anchor are, for example Jurkat, AM-B, C84, BW, S49, CHO and Raji.

3) Interference in or use of a cell deficient in transamidases can be used to inhibit the binding of a GPI anchor to the protein, causing the protein to lack anchoring and be secretable. Said deficient cell has been obtained by mutagenesis in CHO cells.

It is obvious to those skilled in the art that it can be further modified and secreted into a modified phosphatase comprising a crown domain and a catalytic domain, wherein said crown domain and said catalytic domain are obtained from different alkaline phosphatases. Therefore, in a preferred embodiment, the FAL is an isolated or recombinant phosphatase comprising a modification in the glycosylphosphatidylinositol (GPI) signal sequence, wherein said modification produces a secreted phosphatase and wherein said recombinant phosphatase further comprises a crown domain and a catalytic domain that are obtained from different phosphatases. In Figure 10, non-restrictive examples of such mutant (alkaline) phosphatase are provided. Such a combined or "double" mutant produces, for example, a modified phosphatase with a certain specific activity, stability or specificity of the substrate and, at the same time, the production of such a product is much greater due to the fact that it can be isolated from the medium. surrounding the producing cells.

FAL can be administered by different routes, for example, intravenously, rectally, bronchially or orally.

In a preferred embodiment, the route of administration used is intravenous. It is evident to the person skilled in the art that, preferably, an effective amount of FAL is administered. As a starting point, 10500 U / Kg / day can be used. If the intravenous administration route is used, the FAL (at least for a certain period of time) is preferably applied by continuous infusion.

Compositions comprising a source of FAL are disclosed, among which there are pharmaceutical and nutraceutical compositions comprising a source of FAL. The compositions may optionally comprise pharmaceutically acceptable excipients, stabilizers, activators, vehicles, permeators, propellants, disinfectants, diluents and preservatives. Suitable excipients are commonly known in the art of pharmaceutical formulation, and those skilled in the art can easily find and apply them in references such as, for example, "Remmington Pharmaceutical Sciences", Mace Publishing Company, Philadelphia, PA., XVII ed . 1985. Preferably, compositions comprising a source of FAL are suitable for oral administration and comprise an enteric coating to protect FAL from the adverse effects of gastric juices and a low pH. Enteric coating and controlled release formulations are well known in the art (references described above). The enteric shell compositions of the art may be composed of a solution of a water-soluble enteric coating polymer mixed with the active ingredient (s) such as FAL and other excipients, which are dispersed in an aqueous solution and which subsequently they can be dried and / or sedimented. The enteric shell formed offers resistance to the FAL against the attack of atmospheric moisture and oxygen during storage, as well as gastric fluids and a low pH after ingestion, while it easily decomposes in the alkaline conditions that exist in the lower intestinal tract.

An effective amount of FAL is provided to a subject (preferably, a human being) by any suitable route of administration and with the FAL in any appropriate form. Preferably, the parameters indicative of renal function are determined before the administration of the FAL and after the administration of the FAL, which allows to determine if the treatment is successful. The administration of additional doses is repeated as many times as necessary, preferably, until the parameters of renal function are considered acceptable. An example of a suitable parameter is the presence or absence of an RNA molecule. Preferably, said RNA molecule is an mRNA molecule. Even more preferably, said mRNA molecule is iNOS mRNA. Most preferably, said RNA is obtained from renal cells secreted in the urine.

Another preferred mode of administration comprises the use of pharmaceutical compositions comprising sources of FAL that can be administered in a daily dosage schedule for a prolonged period of time. Preferably, the pharmaceutical compositions comprise an enteric coating to protect the FAL from the harmful effects produced by the gastric juices (pH 1.0 to 2.5) and ensure the effective administration of the FAL.

In yet another embodiment, the use of FAL in the preparation of a medicament to improve reduced renal function is combined with any other therapy (i.e. combination therapy). Other therapy of this type is also directed, for example, to improve renal function. Examples of other therapies aimed at improving renal function have been described above. Non-restrictive examples are treatment with furosemide (Lasix), calcium or dialysis. Other examples of suitable combination therapy are treatment with FAL and at least one iNOS inhibitor or treatment with FAL and at least one TNFa inhibitor. The active compounds can be administered sequentially or at the same time.

In yet another embodiment, the invention provides FAL for use in the treatment of reduced renal function, wherein said renal function is reduced as a result of renal failure. The FAL for use according to the invention can be extended further by identifying a subject suffering from reduced renal function. In one embodiment, the invention provides FAL for use in the treatment of reduced renal function, in which the improvement in renal function obtained is not due to the detoxification of LPS by the FAL.

An embodiment provides FAL for a use according to the invention, wherein said reduced renal function is induced or maintained or aggravated by a medicament or a drug and / or a toxin. Said medicament, drug and / or toxin is preferably selected from the group consisting of antibiotics, protease inhibitors, chemotherapeutic agents, anti-inflammatory agents, blood pressure medications, insecticides, herbicides, ethylene glycol, contrast dyes, heavy metals and toxins. bacterial

FAL is also provided for a use according to the invention, wherein said reduced renal function is induced or maintained or aggravated due to acute renal failure. Said reduced renal function, preferably, is induced or maintained or aggravated by intrinsic acute renal failure. In one embodiment, said acute renal failure is induced or maintained or aggravated by a medicament or a drug or a toxin, preferably selected from the group consisting of aminoglycosides, chemotherapeutic agents, contrast dyes, heavy metals and bacterial toxins.

A FAL is also provided for a use according to the invention, wherein said reduced renal function is induced or maintained or aggravated due to intrinsic acute renal failure. Said intrinsic acute renal failure is preferably acute tubular necrosis and / or glomerulonephritis. Preferably, said acute tubular necrosis and / or glomerulonephritis is induced or maintained or aggravated by a medicament or a drug or a toxin, most preferably, selected from the group consisting of aminoglycosides, chemotherapeutic agents, contrast dyes, heavy metals and bacterial toxins

FAL is also provided for a use according to the invention, wherein said reduced renal function is induced or maintained or aggravated by decreased renal blood flow and / or ischemia. Said decrease in renal blood flow and / or ischemia, in one embodiment, is induced or maintained or aggravated by dehydration, heart failure, septic shock, severe blood loss, hypertension, atherosclerosis and / or thrombosis. Preferably, said reduction in renal blood flow and / or ischemia is induced or maintained or aggravated by a medicament or a drug or a toxin, most preferably, selected from the group consisting of angiotensin converting enzyme (ACE) inhibitors. ) and non-steroidal anti-inflammatory drugs (NSAIDs).

FAL is also provided for a use according to the invention, wherein said FAL is a mammalian FAL, preferably, a human FAL. More preferably, the FAL is placental FAL, placenta type FAL, intestinal FAL or hepatic / bone / renal FAL. More preferably, the FAL is recombinant.

The invention will be explained in more detail in the following non-restrictive examples.

Experimental part

Materials and procedures

Example 1- Effect of FAL on renal function of patients with sepsis

Patients

Fifteen patients admitted to the intensive care unit with a diagnosis of reduced renal function were randomized to treatment with FAL (2 FAL: 1 Placebo).

Arterial blood and catheterized urine were taken at several time points between 0 and 24 hours after being included in the study. Urine volumes were recorded and the samples were frozen for the determination of NO metabolites at -80 ° C until they were used for the assay as described above (6). Creatinine and protein were determined by usual clinical chemistry.

Intervention

Bovine intestinal alkaline phosphatase (FAL, AM-Pharma, Bunnik, The Netherlands) was obtained from the calf intestinal mucosa of <6 months of age. Patients who met the necessary requirements received either FAL or placebo (2.5mM Tris-HCl, 2.5mM magnesium chloride, 0.05mM zinc chloride, pH 7.3, with 40% glycerol as a stabilizer) intravenously during 24 h in a 2: 1 ratio. Patients randomized to treatment with FAL received an initial bolus injection of 67.5 U / Kg of body weight administered for 10 minutes, followed by a continuous infusion of 132.5 U / Kg for the remaining 23 h and 50 minutes.

Chemical tests

The total amount of stable NO metabolites, nitrate and nitrite were determined as a measure of the production of NO radicals, using the Griess reaction, according to Moshage et al. (18). Heparinized plasma and urine samples were diluted by four and forty with distilled water, respectively. The amounts of glutathione-S-transferase A1-1 (GSTA1-1) and GSTP1-1 in urine were determined to differentiate between a lesion of the proximal and distal tubular cells, and analyzed in triplicate by ELISA as previously described ( 19, 20).

Determination of iNOS mRNA expression

Urine samples were centrifuged at 700 g for 10 minutes at 4 ° C. RNA was isolated from cell pellets and reverse transcribed into cDNA as described previously (6). Human iNOS and GAPDH were amplified with a previously developed gene expression assay provided by Applied Biosystems (iNOS; Hs00167248_m1, GAPDH; Hs99999905_m1). All experiments were performed in triplicate. The amounts of samples were normalized with respect to the expression of the constituent gene GAPDH.

Example 2- Effect of FAL on gentamicin-induced renal failure

The effects of alkaline phosphatase (FAL) in a model of gentamicin-induced nephrotoxicity in Wistar rats were evaluated. The animals were given gentamicin at 120 mg / kg intramuscularly for seven consecutive days. Urine samples were taken 24 hours a day on day 6 to measure urine volume, electrolytes (Na +, K +), creatinine, N-acetylglucosaminidase (NAG) and proteins. Blood samples were taken in terminal phase on day 7; serum creatinine concentrations, NUS and electrolytes (Na +, K +) were measured. Groups treated with gentamicin (n = 8 per group) were given a slow intravenous injection of vehicle or FAL (100 U / Kg), immediately before the daily dose of gentamicin, followed by another dose every 12 hours for seven days consecutive (total of 14 doses of FAL = 1,400 U / Kg). A control group (n = 8) received an intraperitoneal injection of physiological saline on day 0 and slow intravenous injections of the vehicle using the same dosing schedule twice a day, as indicated for the FAL.

Example 3 - Effect of FAL on cisplatin-induced renal failure

The effects of alkaline phosphatase on renal function of male Wistar rats were evaluated after a single intraperitoneal injection of cisplatin at 7.5 mg / Kg (indicated as day 0). Urine samples were collected 24 hours on day 2 and day 5 to measure urine volume, electrolytes (Na +, K +), creatinine and proteins. Blood samples were taken on day 3 and day 6; serum creatinine concentrations, NUS and electrolytes (Na +, K +) were measured. A slow intravenous injection of vehicle or FAL (200 U / kg) was administered to groups treated with cisplatin (n = 8 per group) 30 min before the challenge test with cisplatin, after which a second dose was administered 12 hours later; the dosage i.v. continued on day 1 (x 2) and day 2 (x 1) for a total of 5 doses (total FAL dose of 1,000 U / Kg). A control group (n = 8) received an intraperitoneal injection of physiological saline on day 0 and slow intravenous injections of the vehicle using the same dosing schedule twice a day, as indicated for the FAL.

Example 4 - Effect of FAL in sepsis patients with renal insufficiency

Patients

Thirty-six patients admitted to the intensive care units of eight independent hospitals were randomized, with diagnosis of sepsis to treatment with FAL (2 FAL: 1 Placebo). Arterial blood and catheterized urine were taken at several time points between 0 and 48 hours after being included in the study. The groups treated with verum and placebo were analyzed as a whole or as subgroups limited to patients who had renal insufficiency, defined as serum creatinine> 150 Imol / l at the start of the study, or who were already undergoing renal replacement therapy. Serum creatinine (by usual clinical chemistry), mortality and the need for renal replacement therapy of the groups were analyzed.

Intervention

Bovine intestinal alkaline phosphatase (FAL, AM-Pharma, Bunnik, The Netherlands) was obtained from the calf intestinal mucosa of <6 months of age. Patients who met the necessary requirements received FAL or placebo (2.5mM Tris-HCl, 2.5mM magnesium chloride, 0.05mM zinc chloride, pH 7.3, with 40% glycerol as a stabilizer) intravenously for 24 h in a ratio of 2: 1. Patients randomized to treatment with FAL received an initial bolus injection of 67.5 U / Kg of body weight administered for 10 minutes, followed by a continuous infusion of 132.5 U / Kg for the remaining 23 h and 50 minutes.

Other examples of suitable models that can be used to better demonstrate the efficacy of alkaline phosphatase in the treatment of reduced renal function are:

(one)

 Acute endotoxin-induced renal failure in rats (J. Nephrol. 2005, 18: 374-381)

Acute renal failure can occur in female Sprague-Dawley rats by intravenous injection of LPS (1 mg / kg of O111: B4 from E. coli, Sigma, Germany). This model is characterized by a reduction in the glomerular filtration rate, a reduction in blood pressure and an increase in NOx excretion.

(2)

 Anti-Thy-1.1 model of experimental mesangial proliferative glomerulonephritis

As described in Jefferson et al. (J. Nephrol 1999; 12: 297-307), anti-rat goat thymocyte serum can be produced by repeated immunizations of a goat with Lewis rat thymocytes (2 x 108 cells per injection). Serum is extracted after the second and third injection, combined and a fraction enriched in IgG is obtained by a caprylic acid procedure. Next, a single intravenous dose of 20 mg per 100 g of body weight is used to induce the disease in male Wistar rats of 180230 g.

(3)

 Cadmium-induced glomerulonephritis

Rats, intoxicated by an i.p. daily for 5 days with 500 μg Cd2 + per kg per day and then left without treatment for 15 days show a reduction in the glomerular filtration rate as described in Jacquillet et al. (Am. J. Physiol. Renal Physiol. 2006, 290: 127-137) .2 0 06; 290: 127-137).

In these experimental models, alkaline phosphatase is administered intravenously in order to prevent, delay, stop or reverse the pathological process. Alkaline phosphatase can be administered before the induction of the disease, or before or after the establishment of the disease. Alkaline phosphatase can be administered only once or, during disease establishment, alkaline phosphatase can be administered several times.

or in the form of a continuous infusion. The alkaline phosphatase will be administered, for example, in the dose range of 10 U / Kg / day to 500 U / Kg / day.

The parameters that measure efficacy can be selected, but not limited to, among the following: inflammatory parameters (infiltration, leukocyte and macrophage activation status, cytokine production, complement activation), oxidative stress (H2O2 production, content of myeloperoxides in the kidney, induction of iNOS, production of NOx, etc.), renal damage (antibody disposition, coagulation, histology) and chemical blood tests, for example, creatinine levels.

Results

Example 1- Effect of FAL on renal function of patients with sepsis

Patients

Fifteen patients were randomized (FAL, n = 10; placebo, n = 5) with reduced renal function over a period of fifteen months.

FAL attenuates renal induction of iNOS

A Q-PCR (quantitative RT-PCR) was used to determine the levels of iNOS mRNA from cell pellets that were isolated from urine samples at the start of the study and at three separate time points during the first 24 hours after the intervention. . The relative expression of iNOS was normalized in healthy control volunteers (n = 4, data not shown) for the mean cycle threshold value (CT) of the GAPDH constitutive gene (CT = 23.6 ± 0.3, delta CT = 12.1 ± 0.1) and was set to 1 as described above (6). The expression of iNOS was induced 42 times more in this group of patients compared to controls, and the administration of FAL reduced this induction by 80 ± 5% in the first 24 h. In contrast, patients treated with placebo had a greater increase in iNOS levels during the first 24 hours after their inclusion in the study (840 ± 85%, Fig. 1A) compared to the initial levels.

The NO metabolites in blood were not significantly different between patients treated with FAL and placebo (data not shown). However, urinary excretion of NO metabolites decreased 80% [-85 to -75] from 227 [166-531] at the start of the study to 41 [28-84] Imol / 10 mmol creatinine (P <0 , 05) after 24 hours of the administration of the FAL. After placebo treatment, the amount of urinary NO metabolites increased further by 70% [45-570] (from 81 [64-419] to 628 [65-1479] µmol / 10 mmol creatinine, P <0 , 05). In addition, the cumulative urinary excretion of NO metabolites was significantly lower in patients treated with FAL (Figure 1B).

FAL attenuates kidney damage

None of the patients required renal replacement therapy during the 28 days of the follow-up period. All patients showed impaired renal function with mild proteinuria as shown in Table 1. The first 24 hours, improved plasma creatinine clearance in 45% [30-180] of patients treated with FAL and deteriorated by 25% [-35 to -15] in patients treated with placebo, as illustrated in Table 1. During the follow-up period, mean plasma creatinine levels significantly decreased in patients treated with FAL, while no significant change was observed in patients treated with placebo (Table 1).

Urinary excretion of both GSTA1-1 and GSTP1-1 was high in all patients, indicating proximal and distal tubular damage, respectively. During the first 24 h, the amount of GSTA1-1 in the urine of patients treated with FAL decreased by 70% [-80 to -50] from 32.7 [11.5-131.1] to 6.5 [5.4-15.7] μg / 10 mmol creatinine (P <0.05) compared to a 200% increase [45-525] in placebo-treated patients (26.9 [15, 2-32.8] to 38.9 [33.0-205.8] P <0.05). Cumulative urinary excretion of GSTA1-1A was significantly lower in patients treated with FAL (Fig. 1C). On the other hand, the urinary excretion of GSTP1-1 tended to increase slightly (from 22.7 [13.6-41.3] at the beginning of the study to 11.9 [8.5-82.5] μg / 10 mmol creatinine at 24 h, P = 0.072) after treatment with FAL. However, for the cumulative urinary excretion of GSTP1-1 no significant differences were observed between the two treatment groups during the first 24 hours of treatment (Figure 1D).

Example 2- Effect of FAL on gentamicin-induced renal failure

Gentamicin at 120 mg / kg x 7 caused severe renal insufficiency in rats manifested by polyuria, proteinuria, reduced electrolyte excretion, increased FEN, enzymes (NAG), elevated serum creatinine and NUS, decreased of creatinine clearance, a measure of glomerular filtration rate, and increased kidney weight.

The two daily injections of FAL for 7 days (100 U / Kg bid x 14), being administered the first dose before the daily administration of gentamicin, caused a reduction in serum creatinine (Fig. 2) and NUS levels ( Fig. 3), as well as an increase in the excretion of creatinine in the urine (Fig. 4). These improvements contribute to the increase in creatinine clearance as a result of FAL treatment (Fig. 5). In this example, it is shown that the FAL, 100 U / kg b.i.d. IV x 7 has protective effects against gentamicin-induced nephrotoxicity in rats, with respect to NUS, Scr CCp and UCr.

Example 3 - Effect of FAL on cisplatin-induced renal failure

7.5 mg / kg i.p. of cisplatin caused reduced renal function, as reflected in the following parameters: polyuria, proteinuria, decreased electrolyte excretion, elevated serum creatinine and NUS, and decreased creatinine clearance, a measure of the rate Glomerular filtration

In this example, it is shown that FAL (200 U / Kg x 5 iv), protects against cisplatin-induced proteinuria in rats (Fig. 6), a measure of tubular damage in this model, and improves creatinine clearance endogenous

Example 4 - Effect of FAL in sepsis patients with renal insufficiency

Patients

Thirty-six patients with sepsis were randomized to a placebo (n = 11) or FAL (n = 25) treatment, and serum creatinine levels, the need for renal replacement therapy and mortality were analyzed. In addition, these results were analyzed in a subgroup (placebo n = 5; FAL n = 11) that presented renal failure at the beginning of the study.

Serum creatinine

At the start of the study and at 12 h, 24 h and 48 h after the intervention, serum creatinine levels were measured. Figure 8 shows that the group of patients with sepsis has serum creatinine levels at the start of the study, which demonstrates reduced renal function. Figure 8 also shows that FAL, but not placebo treatment, is able to reduce serum creatinine levels within 48 hours after the start of treatment. This effect becomes more noticeable (Fig. 9), if only patients who present with renal insufficiency (defined as serum creatinine> 150 μmol / l, or who are already undergoing renal replacement at the start of the study) are included. Therefore, it is concluded that, in this example, FAL is able to improve renal function in patients with sepsis and that the effect is more noticeable in sepsis patients with renal failure.

Renal replacement therapy

Table II shows that of all the patients included in the study, 36% required renal replacement therapy (dialysis) during placebo treatment, while 24% of patients treated with FAL needed such treatment. Of the patients who already had renal failure at the beginning of the study, these percentages were 80 and 27%, respectively. Therefore, the data presented in this example show that FAL treatment is able to reduce the need for dialysis in patients with sepsis who have renal failure.

Mortality

During the 90-day observation period, mortality from all causes of the population studied was 28% (Table II). There was a slight advantage (24% of mortality) in mortality treated with FAL compared to that treated with placebo (36%). However, in sepsis, renal failure is the most common terminal organ failure, represented in this study by the higher mortality in the subgroup that presented renal insufficiency (36% in the

5 renal failure group compared to 28% of all patients). Interestingly, the effect of FAL in reducing mortality in the renal insufficiency group (60% in the placebo group versus 27% in the FAL group) was much more potent.

In this example, it is shown that treatment with FAL (200 U / Kg / 24 h) improves renal function in patients with sepsis who have reduced renal function, which reduces mortality and the need for renal therapy

10 substitute.

Table 1: Renal function

Parameter of renal function

Duration FAL (n = 10) Placebo (n = 5)

Total urine volume (ml)

0-24 h 0-24 h 1,876 (940-2,227) 454 (323-533) 1,470 (1,115-2,775) 447 (414-769)

Protein excretion (mg / day)

Creatinine clearance (ml / min)

Start of the study 24 h 54 (24-84) 76 (25-101) * 80 (77-91) 59 (45-59)

Serum creatinine (Imol / l)

Start of study 1 day 7 days 91 (73-138) 83 (58-135) ## 70 (60-90) ## 99 (86-114) 125 (71-129) 106 (73-141)

Data are expressed as the median (25-75% range). Significantly different compared to the placebo group, * P <0.05, or compared to the start of the study, P <0.01).

Table II: Mortality and renal replacement therapy

n of placebo (%)

n assets (%)  total n (%)

All patients

11 (31) 25 (69) 36 (100)

Mortality

4 (36) 6 (24) 10 (28)

TRS required

4 (36) 6 (24) 10 (28)

Sepsis with renal failure *

5 (45) 11 (44) 16 (44)

Mortality

3 (60) 3 (27) 6 (38)

TRS required

4 (80) 3 (27) 7 (44)

TRS: Substitute Renal Therapy * Serum creatinine � 150 Imol / l at the beginning of the study OR with TRS at the beginning of the study

15 Description of the figures

Figure 1. Expression of renal iNOS and urinary excretion of NO and GST. (A) The expression of iNOS mRNA is given for patients treated with placebo (open bars, n = 4) and those treated with FAL (closed bars, n = 8). The relative expression of the iNOS mRNA was normalized in healthy control volunteers (data not shown) for the mean cycle threshold value (CT) of the GAPDH constitutive gene (CT = 23.6 ± 0.3, delta CT = 12.1 ± 0.1) and was set to

20 1. The levels of (B) NO metabolites were measured; of (C) GSTA1-1 and (D) GSTP1-1 in urine at different times after the intervention in patients treated with placebo (L, n = 5) and with FAL (., n = 10). The urinary excretion of NO and GST metabolites was corrected for creatinine excretion and analyzed by ANOVA with repeated measurements on the complete curve. (A) The data are expressed as the mean ± ETE and (B + C + D) as the median with a 25% interval for the placebo and a 75% interval for the FAL. (*; Significantly different compared to the placebo group, P <0.05).

Figure 2. Serum creatinine levels in control rats, rats with gentamicin-induced nephrotoxicity and rats with gentamicin-induced nephrotoxicity treated with alkaline phosphatase.

Figure 3. Blood urea nitrogen (NUS) levels in control rats, gentamicin rats with gentamicin-induced nephrotoxicity and rats with gentamicin-induced nephrotoxicity treated with alkaline phosphatase.

Figure 4. Serum creatinine levels in control rats, rats with gentamicin-induced nephrotoxicity and rats with gentamicin-induced nephrotoxicity treated with alkaline phosphatase.

Figure 5. Creatinine clearance in control rats, rats with gentamicin-induced nephrotoxicity and rats with gentamicin-induced nephrotoxicity treated with alkaline phosphatase.

Figure 6. FAL significantly reduces protein secretion in urine after nephrotoxicity caused by cisplatin. Simulated treatment: no treatment with cisplatin; control treatment: cisplatin + vehicle; FAL: cisplatin + alkaline phosphatase.

Figure 7. FAL reduces the secretion of protein in urine after nephrotoxicity caused by cisplatin. Simulated treatment: no treatment with cisplatin; control treatment: cisplatin + vehicle; FAL: cisplatin

+ alkaline phosphatase.

Figure 8. FAL decreases serum creatinine in sepsis patients with reduced renal function.

Figure 9. FAL decreases serum creatinine in sepsis patients with renal insufficiency (defined as serum creatinine> 150 μmol / l, or who are already undergoing renal replacement therapy at the start of the study).

Figure 10. Sequences of the four human alkaline phosphatase isoenzymes. Note: these are the mature protein sequences (i.e., without signal sequence), but before adding the GPI anchor and concomitant processing of the C-terminal amino acids except for the chimeric FALs.

References

one.

Hendrix P. G., Hoylaerts M. F., “Nouwen EJ and De Broe ME. Enzyme immunoassay of human placental and germ-cell alkaline phosphatase in serum ”. Clin Chem 1990; 36 (10): 1793-1799.

2.

 Le Du M-H, Millán J. L., "Structural evidence of functional divergence in human alkaline phosphatases." J Biol Chem 2002; 51: 49808-49814.

6. Heemskerk S., Pickkers P., Bouw M. P., Draisma A., van der Hoeven J. G., Peters W. H. et al. "Up-regulation of renal inducible nitric oxide synthase during human endotoxemia and sepsis is associated with proximal tubule injury." Clin J Am Soc Nephrol 2006; 1: 853-62.

18.

 Moshage H., Kok B., Huizenga J. R., Jansen P. L. “Nitrite and nitrate determinations in plasma: a critical evaluation”. Clin. Chem. 1995; 41 (6 Pt 1): 892-6.

19.

Mulder T. P, Peters W. H., Court D. A., Jansen J. B. “Sandwich ELISA for glutathione S-transferase Alpha 1-1: plasma concentrations in controls and in patients with gastrointestinal disorders”. Clin. Chem. 1996; 42 (3): 416-9.

twenty.

 Mulder T. P., Peters W. H., Wobbes T., Witteman B. J., Jansen J. B. “Measurement of glutathione Stransferase P1-1 in plasma: pitfalls and significance of screening and follow-up of patients with gastrointestinal carcinoma”. Cancer 1997; 80 (5): 873-80.

Claims (17)

one.

 Use of alkaline phosphatase (FAL) in the manufacture of a medicament to improve reduced renal function, in which said renal function is reduced due to renal insufficiency.

2.

 Use according to claim 1, wherein said renal function is reduced due to acute renal failure.

3.

 Use according to claim 1 or 2, wherein said reduced renal function is induced or maintained or aggravated by an intrinsic acute renal failure.

Four.

 Use according to claim 3, wherein said intrinsic acute renal failure is acute tubular cell damage.

5.

 Use according to claim 3, wherein said intrinsic acute renal failure is glomerulonephritis.

6.

 Use according to any one of claims 1 to 5, wherein said reduced renal function is induced or maintained or aggravated due to a medicament, a drug and / or a toxin.

7.

 Use according to claim 6, wherein said medicament or drug or toxin is selected from the group consisting of antibiotics, protease inhibitors, chemotherapeutic agents, anti-inflammatory agents, blood pressure medications, insecticides, herbicides, ethylene glycol, contrast dyes, heavy metals and bacterial toxins.

8.

 Use according to any one of claims 1 to 7, wherein said reduced renal function is induced or maintained or aggravated by reduced renal blood flow and / or ischemia.

9.

 Use according to claim 8, wherein said reduced renal blood flow and / or ischemia are induced or maintained or aggravated by dehydration, heart failure, septic shock, severe blood loss, hypertension, atherosclerosis and / or thrombosis.

10.

 Use according to claim 8, wherein said renal blood flow and / or ischemia are induced or maintained or aggravated by a medicament, a drug or a toxin.

eleven.

 Use according to claim 10, wherein said medicament or drug or toxin is selected from the group consisting of angiotensin converting enzyme (ACE) inhibitors and non-steroidal anti-inflammatory drugs (NSAIDs).

12.

 Use according to any one of claims 1 to 11, wherein said FAL is mammalian FAL.

13.

 Use according to any one of claims 1 to 12, wherein said FAL is human FAL.

14.

 Use according to any one of claims 1 to 13, wherein said FAL is placental FAL, placenta type FAL, intestinal FAL or hepatic / bone / renal FAL.

fifteen.

 Use according to any one of claims 1 to 14, wherein said FAL is recombinant.

16.

 Alkaline phosphatase (FAL) for use in the treatment of reduced renal function, in which said renal function is reduced due to renal failure.

17.

 Use of alkaline phosphatase (FAL) in the manufacture of a drug to improve reduced renal function, in which the improvement in renal function obtained is not due to the detoxification of LPS by the FAL.